Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site.

Abstract

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.

• Authors: N A Linderoth, M N Simon, J F Hainfeld, S Sastry
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 14 Pg. 11049-54 (Apr 06 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11148208 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antigens, Neoplasm
• Antigens, Surface
• Heat-Shock Proteins
• Peptides
• Recombinant Proteins
• sarcoma glycoprotein gp96 rejection antigens

Topics

• Antigens, Neoplasm (metabolism, ultrastructure)
• Antigens, Surface (metabolism, ultrastructure)
• Endoplasmic Reticulum (metabolism, ultrastructure)
• Escherichia coli
• Heat-Shock Proteins (metabolism, ultrastructure)
• Humans
• Microscopy, Electron, Scanning
• Mutation
• Peptides (genetics, metabolism)
• Protein Binding
• Recombinant Proteins (metabolism, ultrastructure)